The overall objective of the basic research proposed here is to understand the effects of long-term, multigenerational exposure to high levels of contaminants on natural populations of animals inhabiting Superfund sites. We will employ a fish model species, the Atlantic killifish Fundulus heteroclitus, which have evolved resistance to dioxin-like compounds that act through the aryl hydrocarbon receptor (AHR) at numerous sites. Killifish inhabiting New Bedford Harbor (NBH), MA, a polychlorinated biphenyl (PCB)-contaminated Superfund site, exhibit heritable resistance to altered gene expression and toxicity of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) and other AHR agonists as compared to fish from a reference site, Scorton Creek, MA (SC). We have identified and cloned two distinct AHRs (AHR1 and AHR2), ARNT2, hypoxia-inducible factors (HIFs), and an AHR represser (AHRR) in killifish. The killifish AHR1 gene is highly polymorphic and AHR1 allele frequencies differ between populations of dioxin-sensitive (SC) and dioxin-resistant (NBH) fish. The studies proposed here will build on these previous findings to address ecological and biomedical/mecham'stic questions regarding the impact of chemicals at Superfund sites. The major objectives are 1) to understand mechanisms underlying differential sensitivity to the developmental toxicity of HAHs and PAHs that act through AHR-dependent signaling, and 2) to determine the impact of evolved HAH/PAH resistance on the sensitivity to other environmental stressors. The first objective will be achieved by focusing on population genetic and functional aspects of polymorphisms in AHR-pathway genes (Aims 1 & 2). The second objective will be approached by focusing on responses to environmental hypoxia (Aim 3). Specific Aims are: (1) To compare polymorphism (SNP) and haplotype frequencies at the AHR1, AHR2, AHRR, and ARNT2 loci in dioxin-sensitive and -resistant populations of killifish, (2) To determine the association of specific AHR1, AHR2, AHRR, or ARNT2 variants with the resistance phenotype of individual fish and to determine the functional differences among variants, and (3) To assess the impact of the dioxin-resistant phenotype on sensitivity to environmental hypoxia and determine the mechanistic basis for cross-talk between dioxin and hypoxia signaling pathways. The proposed research will take advantage of a unique opportunity to establish the molecular mechanisms of population-level effects of contaminants on a natural population of vertebrate animals inhabiting a Superfund site.